Hptlc steps

Hptlc steps

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  HIGH PERFORMANCE THIN LAYERCHROMATOGRAPHY (HPTLC) Presented by; Aiswarya . A. T, First year M.Pharm, Department of Pharmacy Practice, Grace college of Pharmacy.
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  Usual concentration ofapplied samples 0.1 to 1 µg / µl for qualitative Analysis and quantity may vary in quantization based on UV absorption 1 to 5 µl for spot and 10 µL for band application. MANUAL , SEMI-AUTOMATIC , AUTOMATIC APPLICATION Manual with calibrated capillaries Semi and auto-application through applicators Applicators use spray on or touch and deliver technique for application. Application of sample and standard
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  Manual Sample Applicator easyapplication performed with disposable capillary pipettes, guided by the capillary holder Semi automatic sample applicator suitable for routine use With the linomat , samples are sprayed onto the chromatographic layer in the form of narrow bands solvent of the sample evaporates almost entirely gives a narrow band of selectable length Automatic Sample Applicator Samples are either applied as spots through contact transfer (0.1-5 micro lit) or as bands or rectangles (0.5->50 micro lit) using the spray on techniques
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  Chromatographic Development Start of development Low solvent consumption Reproduciblepre- equilibrium with Solvent vapour 1. Twin trough chamber
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  2. Automatic developing chamber(ADC) fully automatic and independent of environmental effects The activity and pre-conditioning of the layer , chamber saturation, developing distance and final drying can be pre-set and automatically monitored by ADC
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  Detection, scanning & documentationDetectionand visualization · Detection under UV light is first choice - non destructive · Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length) · Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF · Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution · When individual component does not respond to UV - derivatisation required for detection
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  CHROMATOGRAM IMMERSION DEVICE Dipping technique immersedand withdrawn at a controlled uniform speed HPTLC SPRAYER  a charger and a pump unit with two kinds of spray heads  type A is for spray solutions of normal viscosity, e.g. lower alcohol solutions  type B is for liquids of higher viscosity, e.g. sulfuric acid reagents
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  TLC SPRAY CABINET completeremoval of reagent mist while spraying TLC plates TLC PLATE HEATER homogenous heating across the plate
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  Scanning connected to computer. Thescanner features three light sources: a deuterium lamp , a tungsten lamp and a high pressure mercury lamp. The scanning speed is selectable between 1 and 100 mm/s